RESEARCH ARTICLE Open Access Autoantibodies predate the onset of systemic lupus erythematosus in northern Sweden Catharina Eriksson 1 , Heidi Kokkonen 2 , Martin Johansson 2 , Göran Hallmans 3 , Göran Wadell 4 and Solbritt Rantapää-Dahlqvist 2* Abstract Introduction: Autoantibodies have a central role in systemic lupus erythematosus (SLE). The presence of autoantibodies preceding disease onset by years has been reported both in patients with SLE and in tho se with rheumatoid arthritis, suggesting a gradual development of these diseases. Therefore, we sought to identify autoantibodies in a northern European population predating the onset of symptoms of SLE and their relationship to presenting symptoms. Methods: The register of patients fulfilling the America n College of Rheumatology criteria for SLE and with a given date of the onset of symptoms was coanalysed with the register of the Medical Biobank, Umeå, Sweden. Thirty- eight patients were identified as having donated blood samples prior to symptom onset. A nested case-control study (1:4) was performed with 152 age- and sex-matched controls identified from within the Medical Biobank register (Umeå, Sweden). Antibodies against anti-Sjögren’s syndrome antigen A (Ro/SSA; 52 and 60 kDa), anti- Sjögren’s syndrome antigen B, anti-Smith antibody, ribonucleoprotein, scleroderma, anti-histidyl-tRNA synthetase antibody, double-stranded DNA (dsDNA), centromere protein B and histones were analysed using the AtheNA Multi-Lyte ANA II Plus Test System on a Bio-Plex Array Reader (Luminex 200 ). Antinuclear antibodies test II (ANA II) results were analysed using indirect immunofluorescence on human epidermal 2 cells at a sample dilution of 1:100. Results: Autoantibodies against nuclear antigens were detected a mean (±SD) of 5.6 ± 4.7 years before the onset of symptoms and 8.7 ± 5.6 years before diagnosis in 63% of the individuals who subsequently developed SLE. The sensitivity (45.7%) was highest for ANA II, with a specificity of 95%, followed by anti-dsDNA and anti-Ro/SSA antibodies, both with sensitivities of 20.0% at specificities of 98.7% and 97.4%, respectively. The odds ratios (ORs) for predicting disease were 18.13 for anti-dsDNA (95% confidence interval (95% CI), 3.58 to 91.84) and 11.5 (95% CI, 4.54 to 28.87) for ANA. Anti-Ro/SSA antibodies appeared first at a mean of 6.6 ± 2.5 years prior to symptom onset. The mean number of autoantibodies in prediseased individuals was 1.4, and after disease onset it was 3.1 (P < 0.0005). The time predating disease was shorter and the number of autoantibodies was greater in those individuals with serositis as a presenting symptom in comparison to thos e with arthritis and skin manifestations as the presenting symptoms. Conclusions: Autoantibodies against nuclear antigens were detected in individuals who developed SLE several years before the onset of symptoms and diagnosis. The most sensitive autoantibodies were ANA, Ro/SSA and dsDNA, with the highest predictive OR being for anti-dsDNA antibodies. The first autoantibodies detected were anti-Ro/SSA. * Correspondence: solbritt.rantapaa.dahlqvist@medicin.umu.se 2 Department of Public Health and Clinical Medicine/Rheumatology, Umeå University, SE-901 85 Umeå, Sweden Full list of author information is available at the end of the article Eriksson et al. Arthritis Research & Therapy 2011, 13:R30 http://arthritis-research.com/content/13/1/R30 © 2011 Eriksson et al.; licensee BioMed Central Ltd. This is an o pen acce ss article d istributed u nder t he terms o f the Cre ative Co mmons Attribution License (http://crea tivecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the origina l work is p rope rly cited. Introduction Systemic lupus erythematosus (SLE) is a heterogene ous disease with diverse clinical manifestations and variable severity in individual patients and between different patient populations [1,2]. A typical pathophysiological sign in SLE patients is the production of autoantibodies directed against nuclear antigens, which precede the development of clinical manifestations [3,4]. In particu- lar, antibodies against double-stranded DNA (anti- dsDNA) have been shown t o increase just prior to a diagnosis of SLE [5]. Individuals who d evelop SLE have also been found to gradually fulfill the clinical classifica- tion criteria that are pr eceded by the appearance of associated autoantibodies before diagnosis [6]. Further- more, in patients defined as having undifferentiated con- nective tissue disease, a diagnosis o f SLE was predicted in a 5-year follow -up study on the basis of the presence of anti-dsDNA antibodies [7]. There are several autoimmune diseases that are recog- nised by exhibiting a long preclinical phase during which susceptible individuals who later develop disease can be identified by the presence of autoantibodies [8-11]. The development of a rheumatic disease in asymptomatic mothers expressing anti-Sjögren ’ssyn- drome antigen A (Ro/SSA) and/or anti-Sjögre n’ ssyn- drome antigen B (La/SSB) antibodies, and identified by the birth of a child with a congenital heart block, was found to be relatively common at 48% [12]. In another study, the detection of anti-La/SSB antibodies predated clinical evidence of Sjögren’ssyndromebymonthsand in some cases by years [13]. Furthermore, in a n animal model of SLE, mice immunized with human Ro/SSA developed autoimmunity not only towards this molecule but also towards other immunologically similar mole- cules in a process equivalent to epitope spreading [14]. Thepresenceofantinuclearantibodies(ANAs)was shown to predate the development of SLE in a small study conducted in Finl and [15]. In the study by Arbuckle et al. [3] , the frequency of produ cing at least one SLE-related autoantibody years before diagnosis was high at 88%. ANAs were present in 78% of the cases, anti-dsDNA antibodies were present in 55% and anti- Ro/SSA antibodies were pre sent in 47%. Furthe rmore, the appearance of these antibodies appeared to follow a predictable course [3]. Anticardiolipin antibodies have been found to precede both the diagnosis of SLE and the development of clinical manifestations of thrombosis by a number of years [16]. The aim of this study was to analyse, using multiplex technology, the autoantibodies predating the onset of symptoms of SLE in individuals in a patient population in northern Europe and to relate these autoantibodies to the first recorded symptom of disease. Materials and methods Patients and controls The register of patients with SLE attending the Depart- ment of Rheumatology, University Hospital, Umeå, Swe- den, with a known date of the o nset of symptoms was coanalysed with the registers of the Medical Biobank (Umeå, Sweden) and of the maternity cohort (that is, the record of samples obtained for rubella screening of pregnant women) from northern Sweden. All SLE patients had been evaluated clinically. A total of 38 patients (3 male and 35 female, of whom 37 fulfilled four and one fulfilled only three of the American Col- lege of Rheumatology (ACR) criteria for SLE [17,18]) were identified as having donated blood before the onset of any symptoms of disease. One of the patients also fulfilled the criteria for mixed connective tissue dis- ease [19]. Nineteen of the patients w ere identified from the Medical Biobank (on the basis of plasma withdra- wal), and the other 19 were identified from among the maternity cohort collection (on the basis of sera with- drawal). All individuals in the county of Västerbotten are continuously invited to donate blood samples to the Medical Biobank, the plasma from which is stored at -80°C in a biorepository, and blood samples are drawn from all pregnant women with the sera stored at -20°C. Full details of the conditions f or recruitment and the collection and storage of blood samples have been described previously [10]. A nested 1:4 case-control study was undertaken with the 38 identified individuals, referred to hereinafter as “ presymptomat ic” individuals, and randomly selected controls (n = 152) fro m the same popula tion-based cohorts matched for sex, age and date of blood sampling as well as area of residence. The mean age at the time blood sampling of the individuals who subsequently developed SLE was 36.9 years (age range, 16.8 to 60.2 years) and that of the matched controls was 36.7 years (age range, 17.8 to 62.3 years). The patients’ ages at the time of sampling, the time predating the o nset of symp- toms and diagnosis and the time after sampling u ntil the onset of symptoms are presented in Table 1 for both the Medical Biobank (stratified by sex) and mater- nity cohorts. Samples from three prepatients and six controls, all from the maternity cohort, were no longer available; that is, insufficient sera were available for analysis. The frequencies of nonsmokers, ex-smokers and current smokers among the presymptomatic patients were 47.2%, 26.3% and 26.3%, respectively. The equivalent data are not available for the controls. The study was approved by the Regional Ethics Com- mittee of the University Hospital in Umeå, and al l parti- cipants gave their written informed consent. Eriksson et al. Arthritis Research & Therapy 2011, 13:R30 http://arthritis-research.com/content/13/1/R30 Page 2 of 8 Analysis of autoantibodies Autoantibodies against Ro/SSA (52 and 60 kDa), La/ SSB, dsDNA, ribonucleoprotein (RNP), Smith (Sm), his- tidyl-tRNA synthetase (Jo-1), scleroderma (Scl-70), cen- tromere protein B a nd histones in plasma from 19 presymptomatic individuals and matched controls (n = 76) (Medical Biobank), in sera from 16 presymptomatic individuals and matched controls (n =76)(maternity cohort) and in sera from SLE patients (n = 38) were col- lected during the disease. All autoantibodies were detected using the multiplex AtheNA Multi-Lyte ANA II Plus Test (Zeuss Scientific, Raritan, NJ, USA) and analysed on a Bio-Plex Array Reader (Luminex 200 Lab- map™ system;LuminexCorp.,Austin,TX,USA).The cutoff level for a positive value for each autoantibody recommended by the manufacturer was used, that is, 120 AU/ml for all analytes. Analyses of ANAs were per- formed by indirect immunofluorescence on human epi- dermal cell 2 (HEp-2 cells) slides (Immunoconcept, Sacramento, CA, USA) using 1:100 di luted samples. Analyses of the autoantibodies (ANAs, anti-dsDNA, anti-Ro/SSA, anti-La/SSB, anti-Sm, anti-RNP, anti-Jo-1, anti-Scl-70, anti-centromere protein B a nd antihistone) in the sera of the patients at diagnosis were also under- taken b y the routine clinical immunology laboratory at the Un iversity Hospital. ANAs were analysed by indirect immunofluorescence with HEp-2 cells or rat tissue (in house), anti-dsDNA was analysed on Crithidia luci liae- coated slides (Immunoconcept) and the other autoanti- bodies were analysed either by enzy me-linked immuno- sorbent assay or by immunoblot assay. Statistics Statistical calculations were performed using SPSS for Windows version 17.0 software (SPSS, Inc., Chicago, IL, USA). Continuous data were compared by nonpara- metric analyses with the Wilcoxon signed-rank test for matched pairs (prepatients versus SLE patients) and conditional logistic regression analyses (prepatients v er- sus controls). The relationships between categoric al data (positive versus negative) were compared using c 2 analy- sis or Fisher’s exact test as appropriate. All P values are two-sided, and P ≤ 0.05 was considered statistical signif- icant. P values corrected for the number of comparisons made outside the hypothesis are presented as P cor- rected (P c ). Results Analyses in presymptomatic individuals and controls Of the 35 presymptomatic individuals whose blood sam- ples were availa ble, 22 (63%) had any detectab le autoan- tibodies in their blood before the onset of symptoms, that is, predating disease by a median of 4.2 years (range, 2.1 to 7.9 years). Ten of these patient s expressed one autoantibody, whilst 12 others had two or more autoantibodi es (range, from two to seven). The s ensitiv- ity was highest for ANAs at 45.7% with a specificity of 95%, followed by anti-dsDNA and anti-Ro/SSA antibo- dies , both with a sensitivity of 20% but with specificities of 98.7% and 97.4%, re spectively (Table 2). The sensitiv- ities for the other autoantibodies were between 14.3% and 2.9% at 98% to 100% specificity levels (Table 2). The odds ratio (ORs) for predicting the development of SLE were highest for anti-dsDNA at 18.13 (95% confi- dence interval (95% CI), 3.58 to 91.84), followed by ANAs at 11.5 (95% CI, 4.54 to 28.87) and anti-Ro/SSA antibodies at 8.94 (95% CI, 2.45 to 32.58). The ORs for the other antibodies were between 9.36 and 4.29, although the number of positive individuals was low, that is, up to five. The likelihood ratio (LR) was highest for anti-dsDNA antibodies at 15.38, followed by ANAs with a LR of 9.14. Theautoantibodytypetoappearfirstbeforetheonset of symptoms was anti-Ro/SSA antibody at a mean (±SD) of 6.6 ± 2.5 years. Anti-RNP and antihistones also appeared early at means (±SD) of 5.9 ± 2.5 years and 5.0 ± 1.5 years, respectively, although the number o f positiv e individuals with each antibody was small, that i s, four and five, respectively. The autoantibodies first detectable closest to disease onset were anti-centromere protein B at 0.2 years, anti-Sm at 0.7 years and anti-Scl-70 a t a mean (±SD) of 1.4 ± 0.6 years (Table 3). The number of individuals expressing autoantibodies increased the closer they got to the onset of symptoms, that is, 12 (63%) of 19 individuals had autoantibodies present <5 years before disease onset compared with 8 (50%) of 16 individuals who had autoantibodies present ≥5 years before disease onset. The number of autoantibodies per individual also Table 1 Age at sampling, at onset of symptoms and disease onset and predating time presented as median values (Q1, Q3) Medical Biobank Patient characteristics Females (n = 16) Males (n = 3) Maternity cohort (n = 19) Median age at sampling 50.1 (40.2, 52.3) 50.2 (49.2, 60.1) 24.7 (21.7, 29.0) Median age at symptom onset 52.0 (46.8, 61.2) 52.3 (51.0, 62.1) 31.7 (26.5,39.1) Median age at diagnosis 53.5 (48.0, 62.7) 52.8 (51.1, 63.1) 37.8 (30.2, 43.1) Predating time between sampling and symptom onset 5.28 (1.44, 7.88) 2.03 (1.74, 2.06) 6.74 (3.0, 9.24) Eriksson et al. Arthritis Research & Therapy 2011, 13:R30 http://arthritis-research.com/content/13/1/R30 Page 3 of 8 increased the closer the individual got to the onset of symptoms, particularly during the last 3 years before dis- ease onset; however, this change did not reach statistical significance. The accumulated number of individuals who were positive for each antibody before any symp- toms of disease and after disease onset is i llustrated in Figure 1. In the maternity coh ort, 37.5% had autoantibo- dies predating disease, compared with 94% in females and 100% in males from the Medical Biobank cohort. The number of positive autoantibodies increased with age at the time of blood samp ling (P = 0.001, Pc < 0.01). Those individuals who had autoantibodies predat- ing disease onset were older both at the time of blood sampling and at the onset of symptoms (42.8 versus 28.3 years and 49.3 versus 36.0 years; P = 0.002, Pc< 0.05, and P = 0.005, Pc < 0.05, respectively). The interval between blood sampling and the onset of clinical symp- toms was shorter than it was for those who had no autoantibodies in their presymptom sample; however, this finding was not statistically significantly different (mean 5.2 years versus 6.3 years before symptom onset). Analyses in presymptomatic individuals and at diagnosis of SLE The mean number of autoantibodies present in predis- ease individuals was 1.4 and increased after disease onset to 3.1 (P < 0.0005). In the autoantibody positive presymp- tomatic individuals (n = 22), the mean number of autoan- tibodies was 2.2 before and 3.3 after a diagnosis of SLE (P <0.016,Pc < 0. 1), whi lst among the antibody- negative prep atients (n = 13), the mean number of autoantibodies after diagnosis was 2.8 (P < 0.002, P c < 0.05). The autoantibodies present in relation to symptoms at the onset of disease are presented in Table 4. The patients with serositis (n = 6; four females and two of three males) at the onset of symptoms had higher fre- quencies of autoantibodies than did those with arthritis (n = 20; one of three male s) and skin manifestations Table 2 Sensitivity and specificity of autoantibodies before onset of disease symptoms in individuals who later developed SLE a Presymptomatic individuals Autoantibodies Sensitivity, n (%) Specificity n (%) Controls, n (%) OR 95% CI P value b LR ANA 16 (45.7)*** 95.0 10 (6.7) 11.5 4.54 to 28.87 <0.0001 9.14 dsDNA 7 (20.0)*** 98.7 2 (1.4) 18.13 3.58 to 91.84 <0.0001 15.38 Ro/SSA 7 (20.0)*** 97.4 4 (2.7) 8.94 2.45 to 32.58 <0.0001 5.56 Histone 5 (14.3)** 98.0 3 (2.0) 8.06 1.83 to 35.54 <0.001 7.15 RNP 4 (11.4)** 98.7 2 (1.4) 9.36 1.64 to 53.36 <0.01 8.77 La/SSB 3 (8.6)** 100 0 Jo-1 3 (8.6)* 98.7 2 (1.4) 6.80 1.09 to 42.36 <0.05 6.62 Scl-70 2 (5.7) 99.3 1 (0.7) 8.85 0.78 to 100.51 ns 8.0 Sm 1 (2.9) 100 0 Centromere protein B (2.9) 98.7 1 (0.7) 4.29 0.26 to 70.39 ns 1.54 a 95% CI, 95% confidence interval; b P values were determined by using c 2 test or Fisher’s exact test as appropriate. . * = p < 0.05, **= p < 0.01, ***= p < 0.001 ANA, antinuclear antibody; dsDNA, double-stranded DNA; Jo-1, anti-histidyl-tRNA synthetase antibody; La/SSB, anti-Sjögren’s syndrome antigen B; LR, positive likelihood value; ns, not significant; OR, odds ratio; RNP, ribonucleoprotein; Ro/SSA, anti-Sjögren’s syndrome antigen A; Scl-70, scleroderma 70; Sm, Smith. Table 3 Duration in years of the various antibodies preceding the onset of symptoms and disease a Antibody Number of positive test results Interval between positive test and onset of symptoms, mean (SD) Interval between positive test and diagnosis, mean (SD) Ro/SSA 7 6.6 (2.5) 8.1 (2.3) RNP 4 5.9 (2.5) 7.5 (2.5) Histones 5 5.0 (1.5) 6.5 (1.9) ANA 16 4.1 (0.8) 7.5 (1.2) La/SSB 3 4.0 (1.0) 7.0 (0.6) dsDNA 7 3.6 (1.2) 6.6 (2.0) Jo-1 3 2.4 (1.1) 3.1 (1.1) Scl-70 2 1.4 (0.6) 2.1 (0.9) Sm 1 0.7 1.1 Centromere protein B 1 0.2 6.6 ANA, antinuclear antibody; a Ro/SSA, anti-Sjögren’s syndrome antigen A; dsDNA, double-stranded DNA; Jo-1, anti-histidyl-tRNA synthetase antibody; La/SSB, anti- Sjögren’s syndrome antigen B; RNP, ribonucleoprotein; Scl-70, scleroderma 70; SD, standard deviation; Sm, Smith. Eriksson et al. Arthritis Research & Therapy 2011, 13:R30 http://arthritis-research.com/content/13/1/R30 Page 4 of 8 (n = 11; one male), with t he mean number of autoanti- bodies among th ese patients being 2.5, 1.7 and 0.9, respectively. However, the time interval predating dis- ease was shorter for those with primary symptoms such as serositis (median, 1.9 years) in comparison with those with arthritis (6.7 years) and skin manifestations (4.2 years). In one individual, the symptom preceding the onset of disease was nephritis without any autoantibo- dies detectable when analysed 3.7 years before disease onset, although at onset the patient was ANA- and anti- dsDNA-antibody-positive. There was no association between smoking and autoantibody formation in either the number of autoantibody-positive individuals or the number of autoantibodies present. Figure 1 Graph showing the accumulated number of positive individuals for each antibody. Shown as the percentage predating disease onset in years and after diagnosis of the disease. ANA, antinuclear antibody; SSA, Sjögren’s syndrome antigen A; SSB, Sjögren’s syndrome antigen B; dsDNA, double-stranded DNA; RNP, ribonucleoprotein; histon, histone. Table 4 Autoantibodies predating onset of SLE and presenting symptoms at disease onset a Antibody Arthritis (n = 19) Skin manifestation (n = 11) Serositis (n =6) Haematologic disorder (n =2) Neurologic disorder (n =1) Renal disorder (n =1) ANA 9 4 4 1 0 0 dsDNA 5 1 2 0 0 0 Ro/SSA 3 2 2 0 1 0 Histone 4 1 1 0 0 0 RNP 4 0 1 0 0 0 La/SSB 2 1 0 0 0 0 Jo-1 2 0 2 0 0 0 Scl-70 2 0 1 0 0 0 Sm 1 0 1 0 0 0 Mean number of antibodies/patient 1.6 0.9 2.5 No antibody 7 5 2 1 0 1 a ANA, antinuclear antibody; ds DNA, double-stranded DNA; Jo-1, anti-histidyl-tRNA synthetase antibody; La/SSB, anti-Sjögren’s syndrome antigen B; RNP, ribonucleoprotein; Ro/SSA, anti-Sjögren’s syndrome antigen A; Scl-70, scleroderma 70; Sm, Smith. Eriksson et al. Arthritis Research & Therapy 2011, 13:R30 http://arthritis-research.com/content/13/1/R30 Page 5 of 8 In samples analysed after disease onset but during developmen t of the disease, the concentrations of six of the autoantibodies that were positive in presymptomatic patients, namely, the autoantibodies anti-Jo-1 (n =3), anti-Scl-70 (n =2),anti-RNP(n =2),antihistone(n = 2), a nti-Ro/SSA (n = 1) and anti-centromere protein B (n = 1), decreased to below the cutoff values on the basis of either the multiplex detection kit or routine laboratory protocols. Discussion In this study, we have shown that autoantibody seropo- sitivity preceded the onset of SLE, as defined by ACR criteria, by years. In those individuals who subsequently developed SLE, the number of auto antibodies increased gradually. This could suggest a gradual pathogenic pro- cess over a long period. Our results are consistent with data reported in other prospective studies of asympto- matic individuals who later developed SLE [3], rheuma- toid arthritis (RA) [10,11] or other autoimmune diseases [9]. ANAs were in line with the results presented by Arbuckle et al. [3] in that the most prevalent autoanti- bodies were found in individuals before the onset of symptoms. However, the frequency of the different auto- antibodies predating SLE was lower in our study than the frequencies reported by others [3,20]. This could be expl ained by the longer time predating the onset of dis- ease relative to the lower number of samples. Furthermore, one must consider the ethnic back- ground of the different patient cohorts. All of the indivi- duals included in the present stu dy were from northern Sweden, whilst in the two other studies cited [3,20], 62% were black in both studies, with only 29% and 26%, respectively, being of European background. Anti- extractable nuclear antigen (anti-ENA) antibodies have been found to be more common in Afro-Caribbean and African-American populations than in Caucasians [21-23]. Conversely, the importance of ethnic differences in relation to autoantibodies was not confirmed in another study [24]. Another possible explanation for the lower frequency of detectable autoantibodies in the individuals studied here is that one-half of the samples were sera from pregnant women, in whom the fr equency of autoantibo- dies is known to generally be lower. Also, these donors were younger at the time of blood sampling, and conse- quently the time interval before disease onset for most of the individuals was longer. The samples from the maternity cohort were taken early in pregnancy, which can be of importance when consideri ng that these pre- symptomatic individuals had a lower prevalence of auto- antibodies than the remainder of the patients and also that pregnancy is, partially at least, an immunosuppres- sive state. These individuals were also younger at the time of the collection of blood samples, when the symp- toms started and when the diagnosis of SLE was con- fir med. Their samples had also been stored frozen for a longer time, which should be considered as a factor that could interfere with the ana lyses. After the diagnosis was established, these patients had marginally fewer autoantibodies than the other patients, although not sig- nificantly so. It has long been suggested that autoanti- body formation increases with age [25,26] as was found in the present study. In line with the other studies [3,20], anti-Ro/SSA antibodies were the first to be detected and preceded the onset of SLE by several years, whilst anti-Sm and anti-centromere protein B antibodies appeared closer to the onset of clinical symptoms. Also, as described by Arbuckle et al. [3], anti-dsDNA antibodies appeared at an intermediate time point. Our results differ from those of Arbuckle et al. in the way that ANAs appeared at an intermediate point relative to the onset of clinical symptoms and that anti-La/SSB antibodies appeared closer to the onset of symptoms. This finding is consistent with the hypothesis of a progression due to epitope spreading as previously described both in animal models and in SLE patients [27-29]. The individuals who had serositis as the first symptom had more autoantibodies and a shorter time interval between the positive blood sample and disease onset than other onset symptoms, suggesting that a more ser- ious manifestation in the beginning of the disease is associated with faster disease development and more pronounced epitope spreading.However,wewere unable to show a significant increase in the number of autoantibodies preceding symptom or disease onset, but after the onset of disease the number of antibodies increased significantly. The OR for predicting SLE was highest for anti- dsDNA antibodies, followed by ANAs and the other autoantibodies with lower ORs, but all were within the 95% CI for the OR of anti-dsDNA antibodies. The num- ber of individuals positive for most of the other antibo- dies was small: between two and five. In this study, 6.7% of the population based controls were positive for ANAs at a preset specificity of 95%. However, ANA p ositivity alone in healthy individuals was not regarded as a good predictor of developing con- nective tissue disease [30,31]. Two controls were posi- tive for anti-Jo-1 antibodies and one was positive for anti-Scl-70 antibodies, which are rare autoantibodies. However, because of the limited amounts of sera and plasma available from the Medical Biobank, we were not able to undertake any co nfirmatory analyses for anti- ENA or anti-dsDNA an tibodies using alternat ive techni- ques, which would have been desirable. Eriksson et al. Arthritis Research & Therapy 2011, 13:R30 http://arthritis-research.com/content/13/1/R30 Page 6 of 8 The ENA and chromatin antigens are a part of all autoantigens present in the cell nuclei visualised by ANA analysis using immunofluorescence. In the nucleus, there are many antigens other than ENA or chromatin that cannot be detected by specific methods today. Comparison between multiple assays for autoanti- body detection in SLE has shown variable frequency of, for example, Scl-7 0, with higher frequencies published using the same assay as we used i n this study, suggest- ing a too low cutoff value, at least for Scl-70 [32,33]. In this study, we could not find any difference in auto- antibody formation between smokers and nonsmokers. A signifi cantly higher risk of dsDNA seropositivity was found in current smokers compared with those who had never smoked in a previous study of SLE patients [34]. Smoking has been suggested as an environmental factor involved in the pathogenic development of autoantibo- dies to citrullinated proteins and rheumatoid factor in patients with RA [35]. This study is limited by the availability of stored sam- ples and by not having several s amples collected from the same individual before the onset of symptoms. How- ever, these individuals were patients attending one clinic, where they are followed regularly. The controls used in this study were sampled at the same time as the patients, and their samples were collected, stored a nd analysed in the same way. We have also used a newly intro duced multiplex tech- nique, which is similar to that used by Heinlen et al. [20], thereby making comparison with the previ ous pub- lica tion by Arbuckle et al. [3] more difficult. The multi- plex technology is very suitable, since the amount of serum or plasma required is very small relative to the number of analytes it is possible to detect in any given sample. This is of special benefit when analysing stored serum samples from biobanks, where the volumes stored are limited. Conclusions On th e basis of this study, we conclude that autoantibo- dies against nuclear antigens can be detected several years before the onset of symptoms and SLE diagnosis in individuals who subsequently develop SLE. The high- est sensiti vities were for ANA, Ro/SS A and dsDNA, and anti-dsDNA antibodies had the highest predictive value for SLE. Antibodies against Ro/SS A were the first auto- antibodies detected. Individua ls who had serositis as the first symptom h ad more auto antibodies and a shorter time interval between the positive blood sample and dis- ease onset than other onset symptoms, suggesting that more serious disease manifestation in the beginning of the disease is associated with faster disease development and more pronounced epitope spreading. Abbreviations ACR: American College of Rheumatology; ANA II: antinuclear antibody test II; anti-Sm: anti-Smith antibody; dsDNA: double-stranded DNA; HEp-2: human epidermal cell 2; Jo-1: anti-histidyl-tRNA synthetase antibody; La/SSB: anti- Sjögren’s syndrome antigen B; LR: likelihood ratio; OR: odds ratio; RNP: ribonucleoprotein; Ro/SSA: anti-Sjögren’s syndrome antigen A; Scl-70: scleroderma 70; SLE: systemic lupus erythematosus. Acknowledgements This project was supported by funding from VISARE NORR Fund, Umeå Sweden. Author details 1 Department of Clinical Immunology, Umeå University, SE-901 85 Umeå, Sweden. 2 Department of Public Health and Clinical Medicine/Rheumatology, Umeå University, SE-901 85 Umeå, Sweden. 3 Department of Nutritional Research, Umeå University, SE-901 85 Umeå, Sweden. 4 Department of Virology, Umeå University, SE-901 85 Umeå, Sweden. Authors’ contributions CE analysed and interpreted the data and was involved in drafting the manuscript. HK analysed and interpreted the data and was to some extent involved in drafting the manuscript. MJ contributed to the study design and analysed and interpreted the data. GH and GW contributed to the design of the study and were involved with the supply of the blood samples. SRD designed the study, analysed and interpreted the data and was involved in drafting the manuscript. 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Hanly JG, Thompson K, McCurdy G, Fougere L, Theriault C, Wilton K: Measurement of autoantibodies using multiplex methodology in patients with systemic lupus erythematosus. J Immunol Methods 2010, 352:147-152. 34. Freemer MM, King TE Jr, Criswell LA: Association of smoking with dsDNA autoantibody production in systemic lupus erythematosus. Ann Rheum Dis 2006, 65:581-584. 35. Klareskog L, Alfredsson L, Rantapää-Dahlqvist S, Berglin E, Stolt P, Padyukov L: What precedes development of rheumatoid arthritis? Ann Rheum Dis 2004, 63(Suppl 2):ii28-ii31. doi:10.1186/ar3258 Cite this article as: Eriksson et al.: Autoantibodies predate the onset of systemic lupus erythematosus in northern Sweden. Arthritis Research & Therapy 2011 13:R30. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Eriksson et al. Arthritis Research & Therapy 2011, 13:R30 http://arthritis-research.com/content/13/1/R30 Page 8 of 8 . supply of the blood samples. SRD designed the study, analysed and interpreted the data and was involved in drafting the manuscript. All authors have given their final approval of the version of the. analyse, using multiplex technology, the autoantibodies predating the onset of symptoms of SLE in individuals in a patient population in northern Europe and to relate these autoantibodies to the first. with systemic lupus erythematosus. J Immunol Methods 2010, 352:147-152. 34. Freemer MM, King TE Jr, Criswell LA: Association of smoking with dsDNA autoantibody production in systemic lupus erythematosus.